Biofilms are multicellular communities of bacteria that can adhere to virtually any surface. Bacterial biofilms are clinically relevant, as they are responsible for up to two-thirds of hospital acquired infections and contribute to chronic infections. Troublingly, the bacteria within a biofilm are adaptively resistant to antibiotic treatment and it can take up to 1000 times more antibiotic to kill cells within a biofilm when compared to planktonic bacterial cells. Identifying and optimizing compounds that specifically target bacteria growing in biofilms is required to address this growing concern and the reported antibiofilm activity of natural and synthetic host defence peptides has garnered significant interest. However, a standardized assay to assess the activity of antibiofilm agents has not been established. In the present work, we describe two simple assays that can assess the inhibitory and eradication capacities of peptides towards biofilms that are formed by both Gram-positive and negative bacteria. These assays are suitable for high-throughput workflows in 96-well microplates and they use crystal violet staining to quantify adhered biofilm biomass as well as tetrazolium chloride dye to evaluate the metabolic activity of the biofilms. The effect of media composition on the readouts of these biofilm detection methods was assessed against two strains of Pseudomonas aeruginosa (PAO1 and PA14), as well as a methicillin resistant strain of Staphylococcus aureus. Our results demonstrate that media composition dramatically alters the staining patterns that were obtained with these dye-based methods, highlighting the importance of establishing appropriate biofilm growth conditions for each bacterial species to be evaluated. Confocal microscopy imaging of P. aeruginosa biofilms grown in flow cells revealed that this is likely due to altered biofilm architecture under specific growth conditions. The antibiofilm activity of several antibiotics and synthetic peptides were then evaluated under both inhibition and eradication conditions to illustrate the type of data that can be obtained using this experimental setup.

Staphylococcus aureus Biofilms and their Impact on the Medical Field | InTechOpen, Published on: 2017-03-08. Authors: Fany Reffuveille, Jérôme Josse, Quentin Vallé, et

Recent studies have shown that not only resistance, but also tolerance/persistence levels can evolve rapidly in bacteria exposed to repeated antibiotic treatments. We used in vitro evolution to assess whether tolerant/hyperpersistent Escherichia coli ATCC25922 mutants could be selected under repeated exposure to a high ciprofloxacin concentration. Among two out of three independent evolution lines, we observed the emergence of gyrB mutants showing an hyperpersistence phenotype specific to fluoroquinolones, but no significant MIC increase. The identified mutation gives rise to a L422P substitution in GyrB, that is, outside of the canonical GyrB QRDR. Our results indicate that mutations in overlooked regions of quinolone target genes may impair the efficacy of treatments via an increase of persistence rather than resistance level, and support the idea that, in addition to resistance, phenotypes of tolerance/persistence of infectious bacterial strains should receive considerations in the choice of antibiotic therapies.

The increase of multidrug-resistant (MDR) bacteria has renewed interest in old antibiotics, such as minocycline, that can be active against various MDR Gram-negative pathogens. The elimination of minocycline by both kidneys and liver makes it suitable for impaired renal function patients. However, the drawback is the possible elimination of a high amount of drug in the intestines, with potential impact on the digestive microbiota during treatment. This study aimed to predict the potential activity of minocycline against Enterobacterales in the gut after parenteral administration, by combining in vivo and in vitro studies. Total minocycline concentrations were determined by UPLC-UV in the plasma and intestinal content of piglets following intravenous administration. In parallel, the in vitro activity of minocycline was assessed against two Escherichia coli strains in sterilized intestinal contents, and compared to activity in a standard broth. We found that minocycline concentrations were 6–39 times higher in intestinal contents than plasma. Furthermore, minocycline was 5- to 245-fold less active in large intestine content than in a standard broth. Using this PK-PD approach, we propose a preclinical pig model describing the link between systemic and gut exposure to minocycline, and exploring its activity against intestinal Enterobacterales by taking into account the impact of intestinal contents.